Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma...

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Bibliographic Details
Published inJournal of visualized experiments no. 143
Main Authors McCaig, William D, Deragon, Matthew A, Haluska, Jr, Robert J, Hodges, Alexa L, Patel, Payal S, LaRocca, Timothy J
Format Journal Article
LanguageEnglish
Published United States 18.01.2019
Subjects
Buffers
Cell Fractionation - methods
Cell Membrane - metabolism
Centrifugation - methods
Cytosol - metabolism
Humans
Mitochondria - metabolism
Subcellular Fractions - metabolism
U937 Cells
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Summary:In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma membrane. The process can be scaled to accommodate the needs of researchers, is inexpensive and straightforward. This method will allow researchers to determine protein localization in cells without specialized centrifuges and without the use of commercial kits, both of which can be prohibitively expensive. We have successfully used this method to separate cytosolic, plasma membrane and mitochondrial proteins in the human monocyte cell line U937.
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ISSN:1940-087X
1940-087X
DOI:10.3791/59022